Mutations in the cpxA and cpxB genes of Escherichia coli K12 cause an auxotrophy for isoleucine and valine, though neither gene is a knwon ilv locus. The mutations specifically affect acetohydroxyacid synthetase (AHAS) isozyme I among all of the anzymes required for the synthesis of these two amino acids. The specific aims of the proposed research are to determine the effects of the cpx mutations on the synthesis, stability, intracellular location and function of AHAS I, using a combination of biochemical, immunologic and genetic techniques. Using an antiserum against the catalytic component of AHAS I, the product of the ilvB gene, will examine directly the metabolism of the ilvB gene product in mutant and cpx+ cells. We will also examine the relation between AHAS I activity in crude extracts and amino acid pools to determine whether AHAS I activity per se limits growth of mutant cells in the absence of isoleucine and valine. We will determine the intracellular localization of the ilvB gene product in mutant and cpx+ cells and its relation to the ability of subcellular fractions to catalyze the overall synthesis of isoleucine and valine. We will determine whether the valine sensitivity of AHAS I is a property of the catalytically-active ilvB gene product itself, or whether another protein is necessary to confer valine sensitivity on AHAS I. We will then purify the protein and determine wheter or not it is affected by the cpx mutations. Finally, we will characterize mutations that suppress the effects of the cpx mutations on AHAS I, and we will determine whether or not such mutations can be obtained in the ilvB gene itself. This proposal has gworn out of our ongoing research on the cpx genes of E. coli. cpx Mutations alter a variety of cellular properties, in addition to amino acid synthesis, that can be plausibly attributed to deficiencies in knwon envelope proteins. The mechanism by which these mutations alter isoleucine and valine synthesis will therefore elucidate new aspects of the organization and control of these complex and interesting pathways, as well as serve our long-term goal of understanding the role of the cpx gene products in the synthesis and organization of the bacterial cell envelope.